The role of caveolin-1 and Src kinase in HAdV entry and inflammation.
A. C57BL/6J wild type (WT) and caveolin-1 -/- mice (on the C57BL/6J background) were injected intrastromally with Cy3-labeled (red) HAdV-D37 (105 tissue culture infectious doses) or buffer control, and the corneas harvested for confocal microscopy. Phalloidin (green) co-staining was used to better visualize the cells. Virus entry was apparent by 1 hr post infection in WT mice. In caveolin-1 -/- mice, virus remained mostly membrane bound in the first hour post infection and internalization appeared reduced at 24 hr compared to WT mice. B. Western blot analysis of the cornea from mice infected with HAdV-D37 revealed increasing pSrc at 1 and 24 hr post infection, while expression was minimal in untouched (uninfected) corneas. Caveolin-1 -/- mice did not show increased pSrc even after 24 hr infection. Actin controls are shown in the bottom panel. C. ELISA for CXCL1 chemokine performed on WT and caveolin-1 -/- mice corneas at 24 hr post infection with HAdV-D37. Infected caveolin-1 -/- mice corneas showed approximately 60% less CXCL1 expression as compared to infected wild type corneas (*p=.0001). Mock infected mice corneas did not produce any detectable CXCL1. D. Histology of PP2 or DMSO (control) pretreated corneas at 4 days post infection with HAdV-D37 or buffer control shows a reduction of keratitis with chemical inhibition of Src kinase. E. CXCL1 expression by ELISA of HAdV-D37 infected or mock infected corneas at 16 hr post infection pretreated with the Src kinase inhibitor PP2 (10 μM) or DMSO control demonstrates a significant reduction in chemokine expression due to Src inhibition (*p<0.05).
